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Interleukin-10 signaling in regulatory T cells is required forsuppression of Th17 cell-mediated inflammation

Ashutosh Chaudhry1,*, Robert M. Samstein1,*, Piper Treuting2, Yuqiong Liang1, Marina C.Pils3, Jan-Michael Heinrich3, Robert S. Jack3, F. Thomas Wunderlich4, Jens C. Brüning4,Werner Müller3, and Alexander Y. Rudensky1,**

1Howard Hughes Medical Institute and Immunology Program, Memorial Sloan-Kettering CancerCenter, New York, NY 10065, USA2Department of Comparative Medicine, University of Washington, Seattle, WA 98195, USA3Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT UK4Institute for Genetics, University of Cologne, Cologne Excellence Cluster on Cellular StressResponses in Aging-Associated Diseases (CECAD), Center of Molecular Medicine Cologne(CMMC), 2nd Department for Internal Medicine University Hospital Cologne and Max PlanckInstitute for the Biology of Ageing, D-50674 Cologne, Germany

AbstractEffector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplifythe corresponding type of inflammatory response. Regulatory T (Treg) cells integrateenvironmental cues to suppress particular types of inflammation. In this regard, STAT3, atranscription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10(IL-10), and not pro-inflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with theability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cellsresulted in selective dysregulation of Th17 cell responses and colitis similar to that observed inmice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation byserving as principal amplifiers of negative regulatory circuits operating in immune effector cells.

IntroductionProtective immunity against different classes of pathogens is dependent upon generation ofdistinct types of immune responses mediated and coordinated by T helper 1 (Th1), Th2, andTh17 effector CD4+ T cells. The signals promoting differentiation of naïve CD4+ T cellsinto a particular T helper cell subset are provided by distinct sets of secreted and membranebound cytokines elaborated upon triggering of innate immune sensors of infection (e.g. Toll-like receptors, inflammasomes, RIG-I and MDA5) displayed by antigen presenting cells(APCs). Activation of members of the STAT transcription factor family downstream ofcorresponding cytokine receptors is critical for generation of the effector CD4+ T cell

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NIH Public AccessAuthor ManuscriptImmunity. Author manuscript; available in PMC 2011 October 22.

Published in final edited form as:Immunity. 2011 April 22; 34(4): 566–578. doi:10.1016/j.immuni.2011.03.018.

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subsets. For example, activation of STAT3 downstream of IL-6, IL-23, and IL-21 receptorsis required for efficient generation of highly inflammatory Th17 cells essential for protectiveimmunity against yeast, fungi, and extracellular bacteria (Adamson et al., 2009; Littman andRudensky, 2010; Xu and Cao, 2010). Th17 cells specific for self-antigens and, possibly, forcommensal microbiota have been also implicated in autoimmune diseases such asinflammatory bowel disease, arthritis, and psoriasis (Ahern et al., 2008; McKenzie et al.,2006). In both immunity to infection and autoimmunity, effector CD4+ T cells act foremostas critical amplifiers and “recruiters” of the appropriate types of inflammatory immuneresponses mediated by cells of both the innate and adaptive immune systems (Littman andRudensky, 2010).

In addition to a response mode tailored to protect against a particular type of pathogen, asuccessful immune defense strategy requires intricate negative regulation to restrict hosttissue damage caused by the inflammation. Besides cell-intrinsic down-modulation ofsignaling through antigen-specific and innate receptors, these mechanisms includeelaboration of inhibitory soluble mediators by immune effector cells acting in both anautocrine and paracrine manner. Among these mediators, IL-10, which can be produced bymultiple immune cell types, plays a particularly prominent role in curtailing immune-mediated inflammation in the context of infection, allergy, and autoimmunity (Moore et al.,2001; Saraiva and O'Garra, 2010).

The negative regulation afforded by immune effector cells themselves is complemented bysuppression of inflammatory responses by regulatory T (Treg) cells. These cells,characterized by the expression of the X-chromosome-encoded transcription factor Foxp3,are vital for preventing autoimmunity and immune-mediated inflammation elicited bycommensal microbiota and by pathogens, especially, during chronic infection. Treg celldeficiency resulting from deletion or loss-of-function mutations of the Foxp3 gene causes afatal lympho- and myeloproliferative inflammatory syndrome characterized by massivecytokine storm including sharply increased amounts of Th1, Th2, and Th17 cell cytokines(Fontenot et al., 2003). Likewise, ablation of Treg cells in adult healthy mice leads toaugmented generation of Th1, Th2, and Th17 cells and death within two weeks from highlyaggressive inflammatory lesions in a variety of organs (Kim et al., 2007). Theseobservations suggest a possibility that Treg cells are able to control different types of theimmune response by tailoring their suppressive function to a particular inflammatoryenvironment. In support of this notion, in Treg cells, expression of T-bet and IRF-4,transcription factors involved in Th1 and Th2 cell differentiation, respectively, facilitatesTreg cell-mediated suppression of the corresponding type of response (Koch et al., 2009;Zheng et al., 2009). Along the same lines, Treg-specific deletion of STAT3, a transcriptionfactor necessary for Th17 cell differentiation, results in a fatal Th17 cell-driven colitis(Chaudhry et al., 2009). In Treg cells, activated STAT3 and Foxp3 co-operatively regulate asubset of genes, which likely endows Treg cells with the ability to suppress Th17 cell-mediated inflammation (Chaudhry et al., 2009).

STAT3 can be activated downstream of receptors for several pro-inflammatory cytokinesincluding IL-6 and IL-23, which serve as critical inducers of Th17 cell responses, as well asIL-10, known to restrain Th17 cell-mediated inflammation (Gu et al., 2008; McGeachy etal., 2007; Yen et al., 2006). These observations raise a question as to whether the ability ofTreg cells to suppress Th17 cell-mediated inflammation is imparted upon sensing pro-inflammatory (IL-6 and IL-23) or anti-inflammatory (IL-10) cues. The former scenariowould suggest that Treg cell-mediated suppression represents negative feedback regulationinduced by an inflammatory environment. In contrast, the latter scenario implies that Tregcells amplify negative regulators elicited by immune effector cells. Here, we demonstratethat IL-10R, but not IL-6R or IL-23R was required for Treg cell-mediated suppression of

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spontaneous Th17 cell driven colitis. Thus, in analogy with amplification of effectorimmune responses by distinct types of Th cells, Treg cells act as amplifiers of negativeregulatory circuits of immune effector cells to restrain Th17 cell-mediated inflammation.

ResultsIL-10 elicits potent STAT3 phosphorylation in Treg cells

STAT3 activation elicited by IL-6, IL-23, and IL-21 is indispensable for differentiation ofactivated T cells into Th17 effector cells. In addition to these pro-inflammatory cytokines,STAT3 phosphorylation can be induced by the anti-inflammatory cytokine IL-10, known tooppose Th17 cell responses (O'Shea and Murray, 2008). Our recent studies showed that insuppressive Treg cells, STAT3 plays an essential role for their ability to suppress pathogenicTh17 cell responses (Chaudhry et al., 2009). Thus, we sought to explore whether pro- oranti-inflammatory cytokine signaling confers upon Treg cells the ability to restrain Th17cell-dependent inflammation. Since thresholds and consequences of STAT3 activationelicited by different cytokines differ between various cell types, we first assessed STAT3phosphorylation in response to titrated amounts of IL-6 and IL-10 in conventional naïveCD4+ Foxp3- T cells and Treg cells. Amounts of phosphorylated STAT3 (pSTAT3; Y705)were determined using flow cytometric or immunoblot analyses. We found that stimulationof naïve CD4+ and CD8+ T cells with IL-6 induced markedly higher pSTAT3 as comparedto IL-10. In contrast, IL-10 was a more potent inducer of STAT3 phosphorylation in Tregcells as compared to naïve T cells (Fig. 1A, B and C). The total STAT3 amounts were notchanged upon cytokine treatment. These findings indicate that the pro-inflammatorycytokine IL-6 is more effective in inducing STAT3 activation in naïve T cells, whereas inTreg cells, the anti-inflammatory cytokine IL-10 triggers more efficient STAT3phosphorylation. Furthermore, basal amounts of pSTAT3 in unstimulated T cells and Tregcells following IL-23 treatment were not different (data not shown). Several factors canaccount for these differences including varying surface expression of the correspondingcytokine receptors and altered expression of either positive or negative regulators of STAT3activation. Regardless of the mechanism underlying the contrasting pattern of Treg andnaïve T cell responsiveness to IL-6 and IL-10, these results suggest that Treg cells arepoised to sense IL-10 and that IL-10R on Treg cells plays a pivotal role in their function.

Impaired STAT3 phosphorylation in IL-10R, but not IL-6R-deficient Treg cellsTo directly explore this possibility, we induced Treg-specific ablation of IL-10R and IL-6Rby breeding mice harboring a conditional allele encoding IL-10Rα (Il10rafl) or IL-6Rα(Il6rafl) chain with Foxp3Cre mice (Pils et al., 2010; Wunderlich et al., 2010; Rubtsov et al.,2008). Foxp3CreIl10rafl/fl and Foxp3CreIl6rafl/fl mice were born at an expected Mendelianratio and efficiency of deletion was confirmed by quantitative PCR (Fig. S1). We then testedthe assumption that IL-10R plays a non-redundant role in STAT3 activation in Treg cells byanalyzing pSTAT3 in MACS purified CD4+CD25+ Treg cells isolated from spleens andlymph nodes of healthy 3-4 week-old Foxp3CreIl10rafl/fl or littermate controlFoxp3CreIl10rawt/wt mice. IL-10R-deficient Treg cells showed reduced pSTAT3 ascompared to littermate controls. In contrast, no differences in pSTAT3 amounts wereobserved in IL-6R-deficient as compared to IL-6R-sufficient Treg cells (Fig. 1D). Theseresults indicated that IL-10R signaling is an important contributor to STAT3 activation inTreg cells in accordance with the in vitro analysis of STAT3 phosphorylation in Treg cells.

Mice harboring IL-10R deficient Treg cells developed severe immune-mediated colitisAlthough Foxp3CreIl10rafl/fl mice appeared healthy up to 8-10 weeks of age, these micedeveloped clinical symptoms of immune mediated colitis later in life. At ~13-14 weeks ofage they exhibited failure to thrive, splenomegaly and pronounced enlargement of

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mesenteric lymph nodes (Fig. 2A). Despite unchanged or even somewhat reduced numbersof T cells in the lymph nodes and spleen, we observed a marked increase in activatedCD44hiCD62LloCD4+ T cells in these mice as compared to littermate controls (Fig. 2B-D).Moreover, these mice developed weight loss, rectal prolapse, and colon thickening andsuccumbed to the disease by 18-20 weeks of age (Fig. 2G). These clinical manifestations arehallmarks of inflammatory bowel disease (IBD), which was confirmed by histologicalexamination of the intestinal tissues (Fig. 2H). In particular, the colon of the affected miceexhibited marked thickening of the mucosa with dense lymphoid and lesser neutrophilicinfiltration, crypt loss and abscessation, regions of glandular hyperplasia with irregular glandformation and increased mitotic figures. In the most severely affected mice, theinflammation was transmural extending to the attached mesentery (Fig. 2I). Unlike coliticFoxp3CreIl10rafl/fl mice, where essentially all Treg cells lacked IL-10R, heterozygote femaleFoxp3Cre/WtIl10rafl/fl mice, harboring both IL-10R-sufficient and -deficient Treg cells,remained disease-free (Fig. S2). The heightened activation of CD4+ T cells in the presenceof IL-10R-deficient Treg cells was not due to diminished numbers, rather, the frequency andabsolute numbers of Treg cells were increased in Foxp3CreIl10rafl/fl mice (Fig. 2E and F).Furthermore, the IL-10R-sufficient and -deficient Tregs expressed similar amounts of Foxp3(Fig. 2E) excluding diminished Foxp3 expression as an explanation for the pathology inFoxp3CreIl10rafl/fl mice.

IL-10R deficiency does not impair stability of Foxp3 expression in Treg cellsA recent study suggested that upon adoptive transfer of naive CD4+ T cells and Treg cellsinto lymphopenic hosts, innate immune cell production of IL-10 in the recipients is requiredto maintain Foxp3 expression (Murai et al., 2009). In this study, Treg cells lacking IL-10Rlost Foxp3 expression and thus their suppressor function under inflammatory conditions and,hence, severe colitis commenced in lymphopenic hosts. Since Foxp3CreIl10rafl/fl micedevelop similar pathology it was plausible that the phenotype observed in our studies wasdue to a loss of Foxp3 expression in the absence of IL-10R. Although flow cytometricanalysis did not reveal any difference in Foxp3 amounts on per cell basis betweenFoxp3CreIl10rafl/fl mice and control littermates (Fig. 2E), it was still possible that loss ofFoxp3 expression by some IL-10R-deficient Treg cells was masked by proliferation ofnewly generated Treg cells. To address this important caveat, we employed inducibleablation of the Il10rafl allele in Treg cells using Cre-ERt2 recombinase and assessed Foxp3expression (Rubtsov et al., 2010). For these experiments, we crossed the Il10rafl mice toknock-in Foxp3Cre-ERt2 mice that express a triple fusion protein of the enhanced greenfluorescent protein (eGFP) with Cre recombinase and mutated human estrogen receptorligand-binding domain (ERt2) under control of the Foxp3 gene. These mice were equippedwith a Cre-mediated recombination reporter allele containing a loxP site-flanked STOPcassette followed by a DNA sequence encoding yellow fluorescent protein (YFP) knockedinto the ubiquitously expressed ROSA26 locus (R26Y). Treatment of these mice withtamoxifen induced Cre-ERt2-mediated IL-10R ablation and YFP tagging upon excision ofthe STOP cassette. Genetic labeling of Foxp3-expressing cells with YFP using induciblerecombination avoids the continuous incorporation into the labeled population of cells thattransiently up-regulate Foxp3 and affords assessment of Foxp3 stability in differentiatedTreg cells. Thus, Foxp3Cre-ERt2Il10rafl/flR26Y and littermate control mice were administeredtamoxifen and analyzed 3 and 12 weeks later for Foxp3 expression in YFP+CD4+ T cells.We found that IL-10R ablation did not affect Foxp3 expression within splenic and lymphnode cells and that essentially all YFP+ tagged cells expressed unaltered amounts of Foxp3at both time points (Fig. 3A). These results were also confirmed by intracellular staining forFoxp3 and flow cytometric analysis of FACS-purified YFP+ cells at both time points (Fig.3B). Moreover, analysis of Foxp3 mRNA expression in the sorted YFP+ cells byquantitative PCR (qPCR) also did not detect a measurable loss of Foxp3 in IL-10R ablated

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Treg cells. As expected, expression of IL-10R mRNA were substantially lower in YFP+

Treg cells isolated from Foxp3Cre-ERt2Il10rafl/flR26Y mice as compared to the controlanimals (Fig. 3C). Further characterization of the YFP-tagged IL-10R deficient Treg cellsdid not reveal changes in expression of Th1, Th2 and Th17 specific transcription factors andcorresponding cytokines (Fig. S3). These findings effectively exclude a loss of Foxp3expression as an explanation for the observed inability of IL-10R-deficient Treg cells tosuppress intestinal inflammation.

Unimpaired function of IL-6R and IL-23R-deficient Treg cells in vivoIn contrast to immuno-suppressive effects of IL-10, IL-6 signaling on T cells has beensuggested to be critical for mediating as well as sustaining enteric inflammation (Fasnacht etal., 2009). To identify if IL-6 signaling plays a similar role in Treg cells, we examinedFoxp3CreIl6rafl/fl mice. IL-6R ablation in Treg cells did not result in noticeable changes incytokine production, or activation status and numbers of T cells and other immune cell typesincluding granulocytes, macrophages, B cells, NK cells and dendritic cells (Fig. S4; data notshown). Accordingly, phenotype of Treg cells in Foxp3CreIl6rafl/fl mice wasindistinguishable from that in littermate controls and both groups of mice remained healthy.

To assess potential role for IL-23R in Treg cell function we generated mixed bone marrow(BM) chimeras by transferring T cell-depleted BM cells from IL-23R-deficient (Il23r-/-) or -sufficient Ly5.2 B6 mice mixed with BM cells from Ly5.1+Foxp3- mice into lethallyirradiated (900 rads) Rag2-/- recipients. In these experimental chimeric mice all Treg cellslacked IL-23R expression. Irradiated Rag2-/- recipients reconstituted with Foxp3- BM cellsalone, which served as a positive control for immune-mediated lesions, died within 5-6weeks after BM transfer (data not shown). In contrast, IL-23R-deficient Treg cells appearedfully functional and (Il23r-/- + Foxp3-) → Rag2-/- BM chimeras were healthy and devoid ofmeasurable signs of T cell activation, lymphoproliferation, and immune-mediated pathology(Fig. S5). Thus, in contrast to IL-10R ablation, IL-6R or IL-23R deficiency in Treg cells didnot have noticeable effect on their function.

Increased IL-17 production in the presence of IL-10R-deficient Treg cellsExamination of the surface phenotype of Treg cells from Foxp3CreIl10rafl/fl mice indicated aheightened activation of Treg cells in these mice (Fig. 4A). Although the frequency ofproliferating Ki67+ cells within IL-10R-deficient Treg cell population was unchanged,expression of various characteristic Treg cell surface markers including those associatedwith their suppressor function (eg. CTLA-4) were markedly augmented inFoxp3CreIl10rafl/fl mice in comparison to littermate control mice. The observed Treg cellactivation was due to severe colonic inflammation since IL-10R-deficient Treg cells did notexhibit signs of activation in the presence of IL-10R-sufficient counterparts in healthyheterozygote female Foxp3Cre/WtIl10rafl/fl mice (Fig. S2). Thus, IL-10R-deficient Treg cells,like STAT3-deficient Treg cells, fail to restrain colitis likely due to impaired suppressorfunction.

Recent work has demonstrated that inflammatory Th1 and Th17 responses can bothfacilitate colitis development and progression (Brand, 2009). Therefore, we assessed T cellproduction of different effector cytokines in Foxp3CreIl10rafl/fl mice. We observed elevatedproduction of Th17 cell-associated cytokines, IL-17 and IL-22, by CD4+Foxp3- T cells inthe presence of IL-10R-deficient Treg cells (Fig. 4B and C). In contrast, production of Th1-and Th2-associated cytokines IFN-γ, IL-4, and IL-5 was comparable in Foxp3CreIl10rafl/fl

and littermate control mice. Production of other cytokines such as IL-2 was also unaffected(Fig. 4B and C). Notably, IL-10R-deficient Foxp3+ Treg cells did not produce any of these

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cytokines (Fig. 4B). Thus, IL-10R-deficient Treg cells were capable of keeping Th1 and Th2responses in check.

IL-10R-deficient Treg cells fail to suppress Th17 cell responses in vivoThese results were consistent with the idea that only a distinct aspect of the suppressorprogram was impaired in IL-10R-deficient Treg cells, whereas their general suppressivecapacity was intact. This notion was supported by unimpeded ability of IL-10R-deficientTreg cells to suppress proliferative responses of naïve T cells in vitro (Fig. 5A). To examinesuppressor function in vivo, we co-transferred flow cytometry-purified IL-10R-deficient or -sufficient Ly5.2+ Treg cells and effector Ly5.1+ CD4 T cells from Foxp3- mice into Rag2-/-

recipients. Prior to transfer, the effector CD4+ T cell population was heavily skewed towardsTh1 cells, but also contained Th2 and Th17 polarized cells (Fig. 5B). Effector Foxp3- T cellstransferred alone into Rag2-/- recipients induced fatal systemic multi-organ autoimmunesyndrome associated with lymphoproliferation and increased Th1, Th2 and Th17 cellcytokine production, similar to that observed in unmanipulated Foxp3- mice. Co-transfer ofIL-10R deficient Treg cells and Foxp3-effector T cells resulted only in dysregulated Th17cell responses as evidenced by increased frequencies and absolute numbers of IL-17-producing Ly5.1+ Foxp3- CD4+ T cells in recipient mice (Fig. 5C) and a colitis similar inseverity to that seen in Foxp3CreIl10rafl/fl mice (Fig. 5D). Despite failure to suppress Th17cell-mediated inflammation, IL-10R-deficient Treg cells were able to restrainlymphoproliferation as well as Th1 and Th2 responses (Fig. 5C). In contrast, all autoimmuneand clinical IBD manifestations were abrogated upon co-transfer of IL-10R-sufficient Tregcells with only mild subclinical and non-fatal colitis noted histologically (Figure 5D). Thus,these results suggest that IL-10R-deficient Treg cells are selectively impaired in their abilityto control inflammatory Th17 cell responses and that this impairment leads to fatal colitis.

IL-10R signaling facilitates IL-10 production by Treg cellsAlthough Treg cells employ multiple mechanisms of suppression, IL-10 produced by Tregcells plays an important role in preventing colonic inflammation. Mice with Treg-specificablation of IL-10 develop spontaneous colitis (Rubtsov et al., 2008). Thus, it was plausiblethat IL-10 produced by immune effector cells elicited STAT3 phosphorylation in Treg cells,which in turn induced IL-10 production in these cells as well as other mediators ofsuppression. STAT3 has recently been shown to directly bind to a diverse array of targets(Durant et al., 2010). Thus, we examined STAT3 binding to regulatory elements of the Il10gene in Treg cells. Indeed, we found that STAT3 binds at the Il10 locus in Treg cells (Fig.6A). To test whether IL-10R deficiency in Treg cells impairs their ability to produce IL-10,we examined IL-10 expression at the mRNA level in IL-10R-deficient and -sufficient Tregcells using qPCR. These experiments showed significantly diminished production of IL-10by IL-10R-deficient Treg cells (Fig. 6B). Thus, IL-10R signaling in Treg cells leads toSTAT3-dependent expression of IL-10.

In aggregate, our studies implicate sensing of anti-inflammatory cytokine IL-10, but not pro-inflammatory IL-6 and IL-23, by Treg cells as an essential facilitator of their suppressorfunction. IL-10 produced by diverse immune effector cells induces activation of STAT3 inTreg cells and, thereby, facilitates their ability to suppress Th17 cell responses, whichincludes IL-10 production by Treg cells. These results suggest that Treg cells suppress Th17cell-mediated inflammation by acting as amplifiers of negative regulatory mechanismselaborated by immune effector cells (Fig. 6C).

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DiscussionIL-17- and IL-22-producing Th17 cells mediate protection against mycobacterial, fungal,and bacterial infections. Under physiologic conditions, Th17 cells are found predominantlyin the small and large intestine and associated lymphoid tissues where they facilitateproduction of antimicrobial peptides, enforce integrity of the epithelial barrier, and recruitand activate granulocytes and macrophages to restrain pathogenic bacteria (Weaver et al.,2007). Recent studies demonstrated a critical role of certain components of commensal florain induction of Th17 cell responses. In germ-free (GF) mice devoid of commensal flora,Th17 cells are essentially undetectable, but are prominently present upon colonization of GFmice with a single commensal microorganism, segmented filamentous bacterium (Ivanov etal., 2009). Th17 cells play a prominent role not only in resistance against intestinalpathogens such as Shigella or Citrobacter (Ouyang et al., 2008), but also in autoimmune andinflammatory disorders in the gastrointestinal tract including Crohn's disease, and ulcerativecolitis (Ahern et al., 2008). The preferential generation of Th17 cells in the small and largeintestine associated lymphoid tissues and draining lymph nodes is due to high expression offactors supporting Th17 cell differentiation. These factors include IL-1, TGF-β, and STAT3-signaling inflammatory cytokines IL-6, IL-21 and IL-23 (Korn et al., 2009). A completeblock of Th17 cell differentiation in the absence of STAT3 illustrates a critical role for thelatter three cytokines (Mathur et al., 2007; Yang et al., 2007).

Intestinal homeostasis requires restraint of highly inflammatory Th17 cells by a variety ofcell-intrinsic and extrinsic negative regulatory mechanisms. Among mechanisms limitingTh17 cell responses, another STAT3-signaling cytokine IL-10 plays a prominent role (Gu etal., 2008; Li and Flavell, 2008; McGeachy et al., 2007). IL-10 is a powerful negativeregulator of the immune-mediated inflammation with a broad range of target cell types,primarily of hematopoietic origin (Jankovic et al., 2010). IL-10 has been implicated inlimiting inflammation at environmental interfaces, in particular, in the gut and lung mucosa(Groux and Cottrez, 2003; Sun et al., 2009). Germ-line IL-10 deficiency in mice or inabilityof T cells to produce IL-10 leads to immune-mediated colitis (Roers et al., 2004) that iscompletely dependent on enteric microflora (Stehr et al., 2009) defining a major tissue-protective role for IL-10. Not surprisingly, impaired IL-10 signaling has also beenimplicated in the pathogenesis of IBD and recently mutations in the IL-10R genes have beendescribed in two un-related families with a heritable and poorly treatable form of Crohn'sdisease (Glocker et al., 2009).

Multiple immune effector cells, including T cells, B cells, macrophages, dendritic cells, NKcells, neutrophils, eosinophils, and mast cells, express IL-10 (Saraiva and O'Garra, 2010). Ina naïve mouse, B cells have been reported to constitute a dominant source of IL-10 inseveral lymphoid tissues and B cell-derived IL-10 plays a non-redundant role in restrainingT cell responses (Madan et al., 2009). Unlike several other cell types where IL-10 plays animmunosuppressive function, IL-10 on B cells acts to prevent apoptosis, enhancesproliferation, differentiation, and MHC II expression in addition to playing a role inregulation of Ig class switching (Sabat et al., 2010). The differential activation of STAT3observed in B cells upon IL-10 as opposed to IL-6 stimulation probably reflects the crucialrole of IL-10 in B cell function.

Within the T cell subset, activated CD8+ T cells and all three main effector CD4+ T celltypes, Th1, Th17, and Th2 cells are capable of secreting IL-10 (Li and Flavell, 2008). Thus,IL-10 production represents an essential negative feedback mechanism elaborated byimmune effector cells to limit their activation.

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In addition to immune effector T cells, suppressive Foxp3- Tr1 and Foxp3+ Treg cellsproduce IL-10 in the small and large intestine. Recently, we demonstrated that expression ofSTAT3 in Treg cells is a prerequisite for their ability to suppress Th17 responses and thatablation of STAT3 in Treg cells results in fatal Th17 cell-mediated colitis (Chaudhry et al.,2009). Since STAT3 is required for both elaboration of inflammatory Th17 cell responsesand for their suppression by Treg cells, two models of Treg cell-mediated control of Th17inflammatory responses can be considered. First, Treg cells directly sense the inflammatoryenvironment favoring Th17 cell differentiation, i.e. high amounts of IL-6 and IL-23, whichactivate STAT3 and, thereby, enable Treg-mediated suppression of Th17 cell-dependentinflammation. Alternatively, Treg cells respond to the anti-inflammatory mediator IL-10produced by the immune effector cells. According to this scenario, Treg cells act asamplifiers of the negative regulatory mechanisms elaborated by immune effector cells inanalogy to the aforementioned amplification of immune responses by effector T cell subsets.Our finding that IL-10R signaling in Treg cells was necessary for their suppression of Th17cell responses, whereas IL-6R or IL-23R was dispensable strongly argues in favor of thelatter possibility. In addition, this finding suggests that the essential role for STAT3 in Tregcells for suppression of Th17 cell inflammation is dependent upon its phosphorylationdownstream of cytokine receptors and subsequent changes in gene expression.

Colitis observed in Foxp3CreIl10rafl/fl mice was similar to that observed in mice harboringSTAT3-deficient Treg cells suggesting that IL-10 is the predominant activator of STAT3signaling in Treg cells. Like STAT3-deficient Treg cells, IL-10R deficient Treg cells alsofail to suppress Th17 cell responses, whereas no increases in Th1 or Th2 cell cytokines werenoted. Nevertheless, clinical colitis progression in the presence of IL-10R-deficient Tregcells was slower than that in the presence of STAT3-deficient Treg cells implying that anadditional STAT3 signaling cytokine(s) can contribute to some extent to STAT3 activationin Treg cells.

Recent work has suggested that Treg cells may lose Foxp3 expression in inflammatorysettings and these so called “ex-Treg” cells can contribute to pathology (Murai et al., 2010;Zhou et al., 2009). In particular, IL-10 signaling was shown to be necessary for Treg cellstability in an experimental colitis model. A prominent loss of Foxp3 was observed upontransfer of purified Il10rb-/- Treg cells together with naive CD4+CD45RBhi T cells intolymphopenic recipients (Murai et al., 2009). However, we found similar Foxp3 proteinexpression in IL-10R-deficient and -sufficient Treg cells and increased numbers of Foxp3+

cells were present in Foxp3CreIl10rafl/fl mice. Furthermore, we failed to detect loss of Foxp3expression upon co-transfer of effector Foxp3- cells and IL-10R-deficient Treg cells intolymphopenic recipients. Finally, we did not observe detectable loss of Foxp3 expressionupon inducible deletion of IL-10R in Treg cells combined with the cell fate mappingapproach using Foxp3Cre-ERt2 and R26Y recombination reporter allele (Rubtsov et al., 2010).These results indicate that despite loss of IL-10 signaling, Treg cells remain stable andpotential down-regulation of Foxp3 cannot explain the inflammatory colitis observed withTreg-specific deletion of IL-10R. One possible explanation for discrepant results is that fewcontaminating Foxp3- cells can rapidly expand in lymphopenic settings (Komatsu et al.,2009) and this expansion in numbers can be further exacerbated by a lack of IL-10signaling. Furthermore, these incongruent results can also be potentially dependent onwhether Il10ra or Il10rb was utilized to inactivate IL-10 signaling. Whereas IL-10Rα,required for ligand binding, is unique to the IL-10R complex, IL-10Rβ, essential to mediatedownstream signaling, is shared between several members of the class II cytokine receptorssuch as IL-22R and IFN-λR family members (Pestka et al., 2004). Although the expressionand function of these receptors on Treg cells have not been characterized, it remains possiblethat functions of IL-10Rβ unrelated to IL-10 signaling can account for this discordance.

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Although Treg cell-mediated suppression is not fully understood in mechanistic terms,various studies indicate that Treg cells utilize multiple means to accomplish suppression ofdifferent inflammatory responses (Shevach, 2009; Vignali et al., 2008). Our observation thatphosphorylated STAT3 binds to regulatory elements of the Il10 gene in Treg cells and thatIL-10R deficiency in these cells is associated with drastically reduced IL-10 expression isindicative of a feed-forward IL-10 loop operating in Treg cells, i.e. IL-10 produced byvariety of cellular sources augments its own production by Treg cells. Importantly, ablationof IL-10 in Treg cells leads to spontaneous colitis albeit less severe than that inFoxp3CreStat3fl/fl or Foxp3CreIl10rafl/fl mice (Rubtsov et al., 2008). Furthermore, impairedIL-10 signaling in effector T cells results in augmented Th17 cell, but not Th1 cell responsesand Treg cells can restrain these Th17 cell responses in an IL-10 dependent manner (Huberet al., 2011). These observations corroborate our findings that reduced IL-10 production byIL-10R deficient Treg cells is likely to affect Th17 but not other T cell responses.

Our studies indicate that Treg cell-mediated suppression of Th17-driven pathology isfacilitated by activation of STAT3 downstream of IL-10R engagement by IL-10. Subsequentinduction of a STAT3-dependent Th17-suppression program including IL-10 production byTreg cells can then result in dampening of this Th17 response. However, it is also possiblethat Treg cells can act on effector T cells to induce IL-10 expression (Kearley et al., 2005).In either scenario, we suggest that Treg cells can act as amplifiers of negative regulatorymechanisms, such as IL-10, that are initially elaborated by immune effector cells upon theiractivation. This is in analogy with amplification of a particular type of innate and adaptiveimmune responses by a given effector CD4+ T cell type.

MethodsAnimals

Foxp3-, Foxp3Cre, Foxp3Cre-ERt2, Rosa26YFP, Il6rafl and Il10rafl mice have been previouslydescribed (Fontenot et al., 2003; Pils et al., 2010; Rubtsov et al., 2010; Rubtsov et al., 2008;Wunderlich et al., 2010). IL-23R-deficient mice were kindly provided by Vijay Kuchroo(Harvard Medical School). C57BL/6J were purchased from the Jackson Laboratories (BarHarbor, ME). All the mice were bred and housed in the specific pathogen-free animalfacility at the Memorial Sloan-Kettering Cancer Center and used in accordance withinstitutional guidelines. Tamoxifen (Sigma) was dissolved in olive oil (Fluka) to a finalconcentration of 40mg/ml. Mice received three doses of tamoxifen (8 mg each) at days 0, 1,and 3 by oral gavage.

Reagents, flow cytometry and cell isolationFluorophore-conjugated antibodies were purchased from BD-Biosciences and eBioscience.Stained cells were analyzed using a FACSCanto or LSRII flow cytometer (BD Biosciences)and data were analyzed using FlowJo software (Treestar). Intracellular Foxp3 staining wasperformed using Foxp3 mouse Treg cell staining kit (eBioscience). Flow cytometric analysisof pSTAT3 staining was performed according to manufacturer's instructions by immediatefixation using BD Phosflow Fix Buffer and Perm Buffer III (BD Biosciences). Cytokinestaining was performed after stimulation of splenocytes with PMA (50ng/ml) and Ionomycin(500ng/ml) for 4-6 h in the presence of Golgi-Plug (BD Biosciences). All antibodies usedfor flow cytometry staining were purchased from eBioscience or BD Biosciences.

Single-cell suspensions from lymph nodes and spleen were generated by mechanicaldisruption. CD4+CD25- naïve T cells and CD4+CD25+ regulatory T cells were purifiedusing MACS isolation kits (Miltenyi Biotec, Germany) according to the manufacturer's

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protocol. Foxp3-YFP+ and Foxp3-YFP- cells were isolated by sorting on a FACS Aria cellsorter (BD Biosciences).

In vitro suppression assaysFor in vitro suppression assays, Treg cells and effector T cells were purified by positiveselection using CD4-specific beads (Invitrogen) followed by FACS sorting. Antigen-presenting cells (APC) were prepared from wild-type C57BL/6J splenocytes by T celldepletion using Thy1-specific MACS beads. Effector T cells (2×104 cells/well) were co-cultured with Treg cells at indicated ratios in the presence of irradiated (2000 rad) APCs(1×105 cells/well) in 96-well plates in the presence of CD3 antibody (1 μg/ml) for 60 h andpulsed with 1 μCi 3H-thymidine for additional 8-12 hr. Cell proliferation was measuredusing a scintillation counter. The data are presented as mean cpm 3H-thymidineincorporation in triplicate cultures and standard deviation.

Western blot analysisNuclear extracts from MACS (Miltenyi)-purified CD4+CD25- and CD4+CD25+ T cells orFACS-sorted CD4+Foxp3- and CD4+Foxp3+ T cells were prepared using nuclear lysisbuffer (Active Motif) according to the manufacturer's protocol and subjected to western blotanalysis with antibodies specific for STAT3 and pSTAT3 (Y705) (Cell SignalingTechnologies).

Quantitative PCRTotal RNA was extracted with TriZOL reagent (Invitrogen) from FACS purified IL-10R-sufficient and -deficient CD4+YFP+ Treg cells. cDNA was synthesized using SuperscriptIIIReverse Transcriptase (Invitrogen), followed by real-time PCR analysis (SYBR green;Applied Biosystems).

Histopathological evaluation of colitisSamples of cecum and colon from 3-5 animals per group were fixed in 10% neutral bufferedformalin and routinely processed for hematoxylin and eosin staining. Histological evaluationof colitis was performed as described previously (Burich et al., 2001). In brief, the cecum,proximal, middle and distal colon were scored on a 0-4 scale each for mucosal changes(erosion, ulceration, and/or hyperplasia), inflammation, and extent of section involvementby a pathologist. The total inflammation score (IBD score) was derived by summing thescores from the individual segments and subsequently, the mean was derived for eachexperimental group.

Chromatin immunoprecipitationChIP was performed as described previously (Zheng et al., 2007). CD4+CD25+ Treg cellsfrom 6-8 month old mice were isolated using Treg isolation kit (Miltenyi Biotec). Antibodyto STAT3 was used from Cell Signaling Technology. Relative abundance of regions ofinterest in precipitated DNA was measured by qPCR using Power SYBR-Green PCR mastermix (Applied Biosystems).

Statistical MethodsUnless otherwise noted statistical analysis was performed using the Student's t test onindividual biological replicates in Prism (GraphPad).

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

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AcknowledgmentsWe would like to thank Antonio Bravo, Jennifer Herlihy, Julia Gerard and Payam Zarin for help with the mousecolony management, Hana Lee for superb technical assistance, Jean-Christophe Renauld for anti-IL-22, VijayKuchroo for Il23r-/- mice, Dominik Schenten, Simone Nish and Ruslan Medzhitov for help in facilitating keyexperiments. This work was supported by grants from the National Institutes of Health (A.Y.R). A.C. is supportedby the Irvington Institute Fellowship Program of the Cancer Research Institute. R.M.S. was supported by NIHMSTP grant GM07739. A.Y.R. is an investigator with the Howard Hughes Medical Institute.

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Figure 1. IL-10 induces robust STAT3 phosphorylation in Treg cells(A) Flow cytometric analysis of pSTAT3 (Y705) amounts after treatment of splenicCD4+Foxp3- T cells, CD4+Foxp3+ Treg cells and CD8 T cells with recombinant IL-6 (16ng/ml) or IL-10 (100 ng/ml) for 15 mins. Analysis of pSTAT3 levels by either flowcytometry (B) or western blotting (C) after stimulation of indicated cells with titrating doses(3, 10 and 15 ng/ml for IL-6; 50, 150 and 300 ng/ml for IL-10) of recombinant IL-6 orIL-10. (D) Immunoblot detection of pSTAT3 and STAT3 in Treg cells isolated from IL-10Ror IL-6R deficient and sufficient mice. Data are representative of four independentexperiments.

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Figure 2. Treg cell-specific IL-10R deletion results in severe intestinal inflammation(A, B) Spleen and lymph node cellularity in Foxp3CreIl10rafl/wt and Foxp3CreIl10rafl/fl mice(** p<0.001; * p<0.01). (C, D) Frequencies of activated T cells (E, F) Absolute numbers ofCD4+Foxp3+ T cells and (G) body weights of Foxp3CreIl10rafl/fl mice (n=7). (H) IBDscores derived from evaluation of colon and cecum from 14-16 week-old Foxp3CreIl10rafl/wt

and Foxp3CreIl10rafl/fl mice (n=4/group). (I) Representative H&E stained sections of largebowel at the level of the ileocecal-colic junction from Foxp3CreIl10rafl/wt andFoxp3CreIl10rafl/fl mice. Note: Mucosa (M) with expanded irregular, hyperplastic crypts,glandular loss and fibrosis with multifocal transmural inflammation (arrow). Lumen (L).Original magnification, 5x (n=7).

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Figure 3. IL-10R ablation does not affect Foxp3 expression and Treg cell lineage stability(A) Flow cytometric analysis of Foxp3 and YFP expression on splenic CD4+ T cells inFoxp3CreIl10rafl/flR26Y and control littermate mice. (B) Expression of Foxp3 in flowcytometry-sorted CD4+YFP+ T cells from indicated mice. (C) Quantitative PCR (qPCR)analysis of Foxp3 and IL-10Rα expression in flow cytometry-sorted CD4+YFP+ T cells. Thegrey bar represents CD4+YFP- T cells. The results represent the mean+/-SD of relativeexpression values for the indicated genes relative to hypoxanthine-guanine phosphoribosyltransferase. Data are representative of three independent experiments.

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Figure 4. Increased Treg cell activation and selective dysregulation of Th17 cell responses inFoxp3CreIl10rafl/fl mice(A) Flow cytometric analysis of Treg cell activation markers and putative effector moleculeson splenic CD4+Foxp3+ T cells in Foxp3CreIl10rafl/fl and control littermate mice. (B, C)Flow cytometric analysis of cytokine production by splenic CD4+Foxp3- T cells inFoxp3CreIl10rafl/wt and Foxp3CreIl10rafl/fl mice after PMA and Ionomycin treatment for 4-6hours (** p<0.001; * p<0.01).

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Figure 5. IL-10R-deficient Treg cells efficiently control in vitro T cell proliferation but fail tosuppress Th17 cell differentiation and colitis(A) IL-10R-deficient and -sufficient Treg cell-mediated suppression of effector T cell (TEff)proliferative responses in vitro. (B) Frequencies of Th1, Th2 and Th17 skewed cells amongthe transferred T cell population isolated from the Foxp3- mice. (C) Frequencies of splenicTreg cells, CD4+IL-17+Foxp3- T cells, CD4+IFN-γ+ Foxp3- T cells, and CD4+IL-4+Foxp3-

T cells within the indicated donor-derived population 5-6 weeks after co-transfer of eitherFoxp3CreIl10rafl/wt or Foxp3CreIl10rafl/fl Treg cells with Foxp3- CD4+ T cells into Rag2-/-

recipients. (D) Representative H&E stained sections of colon collected from recipient mice5-6 week after adoptive T cell transfer (original magnification, 10x). Note: Mucosa (M),submucosal edema (E), dilated lymphatics (*) and lumen (L).

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Figure 6. IL-10R signaling is required for IL-10 expression by Treg cells(A) qPCR analysis of STAT3-bound chromatin isolated from wild type Treg cells usingprimers corresponding to the indicated genes. House-keeping gene Gmpr was used as aspecificity control while IgG-immunoprecipitated chromatin was used as a negative control.(B) qPCR analysis of relative expression of indicated genes in Treg cells isolated from eitherFoxp3CreIl10rafl/wt or Foxp3CreIl10rafl/fl mice. The results represent the mean+/-SD ofrelative expression values for the indicated genes relative to hypoxanthineguaninephosphoribosyl transferase.

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